Testololactone derivatives



United States Patent 3,083,212 TESTOLULACTGNE DERIVATIVES RichardWilliam Thoma, Somerville, and Josef Fr ed, Princeton, NJ, assignors toOlin Mathieson Chemical Corporation, New York, N.Y., a corporation ofVirginia No Drawing. Filed Nov. 10, 1961, Ser. No. 151,433 4 Claims.(Cl. 260-6432) This invention relates to the production of 16-keto-Atestololactone, 16u-hydroxy-A -testololactone and esters of the latterwtih hydrocarbon carboxylic acids having less than ten carbon atoms.

16a-hydroxy-A -testololactone is prepared by subjecting A-testololactone to the action of the enzymes of the microorganismStreptomyces roseochromogenes under oxidizing conditions, the oxidationbeing effected either by including the starting material in an aerobicculture of the microorganism, or by bringing together, in an aqueousmedium, the substrate, air and enzymes or non-proliferating cells of themicroorganism.

In general, the conditions for culturing Streptomyces roseochromogenesfor the purposes of this invention are (except for the inclusion of thecompound to be converted) the same as those for obtaining cultures ofvarious other actinomycetes for the production of antibiotics and/ orvitamin B i.e., the microorganism is aerobically grown in contact with asuitable fermentation medium. A suitable medium essentially comprises asource of nitrogen and a source of carbon and energy. The latter may bea carbohydrate (such as sucrose, molasses, glucose, maltose, starch ordextrin), a fatty acid, a fat and/ or the starting material itself.Preferably, however, the medium includes an assimilable source of carbonand energy in addition to the compound to be converted.

The source of nitrogenous factors may be organic (e.g., soybean meal,corn steep liquor, meat extract and/or distillers solubles) or synthetic(i.e., composed of simple, synthesizable organic or inorganic compoundssuch as ammonium salts, alkali nitrates, amino acids or urea).

An adequate, sterile air supply should be maintained duringfermentation, for example, by the conventional methods of exposing alarge surface of the medium to air, or by utilizing submerged aeratedcultures. The substrate may be added to the culture during theincubation period, or included in the medium prior to sterilization orinoculation. The preferred (but not limiting) range of concentration ofthe substrate in the culture is about 0.01 to 0.10%. The culture periodmay vary considerably, the range of about 6 to 96 hours being feasible,but not limiting.

This process yields 16a-hydroxy-A -testololactone.

16a-hydroxy-A -testololactone forms esters with organic hydrocarboncarboxylic acids of less than ten carbon atoms, e.g., the lower alkanoicacids as exemplified by acetic, propionic and enanthic acid, the loweralkenoic acids, the aralkanoic acids as exemplified by a-toluic andB-phenylpropionic, the cycloalkane carboxylic acids, the cycloalkanecarboxylic acids, and the aromatic acids as exemplified by benzoic ando, m, or p-toluic acid. The esters of 16u-hydroxy-A -testololactone areprepared by treatment of l6a-hydroxy-A -testololactone with the acidanhydride or acyl halide containing the desired acid group in an organicsolvent (preferably an organic base such as pyridine).

l6a-hydroxy-A -testololactone may be converted to l6 keto-A-testololactone by oxidation, e.g. by treatment with chromium trioxidein the presence of sulfuric acid.

The compounds of this invention have protein anabolic activity and areuseful in the treatment of underweight patients to effect the rapidbuild-up of protein stores.

Patented Mar. 26, 1963 'ice They may be administered orally inconventional oral dosage forms.

The following examples are illustrative of the invention.

EXAMPLE I 16a-Hydroxy-A -Testololactone Glucose g 10 Yeast extract g 2.5K HPO g l Agar g 20 Distilled water to 1 liter is suspended in 5.0 ml.of an 0.01% Duponal (wetting agent) aqueous solution. One ml. portionsof the suspension from two or more slants are used to inoculate ten 250ml. conical flasks, each containing 50 ml. of the following sterilizedmedium B:

Soybean meal g 15 Glucose g 10 Soybean oil g 2.2 CaCO g 2.5

Distilled water to 1 liter.

After 69 hours incubation at 25 with continuous rotary agitation (280cycles per minute; 2 inch radius), 10% (VOL/vol.) transfers are made to79 conical flasks each containing 50 ml. of freshly sterilized medium B.immediately after inoculation, A -testololactone is added to each flaskas 0.25 ml. of a sterile solution containing 12.5 mg. of the compound inN,N-dimethylformamide. A total of 987 mg. is used. After 50 hours offurther incubation, the contents of the flasks are pooled and the pHadjusted to 4.0 With about 15 ml. of l2NH SO The broth is filteredthrough a Seitz clarifying pad using Hy-Flo Filter Aid. The flasks arerinsed with 400 ml. of water, the cake is reslurried, filtered and thefiltrates combined to give about 3500 ml.

(15) Isolation and characterization-The combined filtrate and washingsare extracted four times with 800 ml. portions of chloroform. Thecombined chloroform extracts are filtered and evaporated to dryness invacuo. The residue is taken up in 100 ml. of methanol and an equalvolume of hexane. After thorough shaking and separation of the layers,the methanol extract is freed from methanol, the residual aqueoussuspension extracted with chloroform, the chloroform extract dried oversodium sulfate and evaporated to dryness in vacuo. The crude residue(991 mg.) is triturated with acetone-hexane to yield 464 mg. of16u-hydroxy-A -testololactone, M.P. 196-198 2l3214 C.; [04 68 (C, 0.53in CHC1 kg}; 240 m (e=15,600); hm? 3.05, 5.81, 6.06, 6.21, 6.27, and1128, 1.

Analysis.Calcd. for C H O (316.38): C, 72.12; H, 7.65. Found: C, 72.07;H, 7.56.

EXAMPLE II 1dot-Acetoxy-A -Testololactone chlf.); AW 5.74., 5.80, 6.01,6.18, 6.24am 8.05

max.

Analysis.Calcd. for C H O (358.42): C, 70.37; H, 7.31. Found: (3, 70.81;H, 7.07.

To a solution of 1 g. of l6pi-hydroxy-A -testololactone in 100 ml. ofpure acetone is added slowly with stirring 1.4 ml. of a solution of 200mg. of chromium trioxide and 320 mg. of concentrated sulfuric acid permilliliter of water. After 35 minutes a few drops of ethanol are addedfollowed by 50 ml. of water. The acetone is evaporated in vacuo and theaqueous suspension extracted with chloroform. The chloroform extractis'washed with sodium bicarbonate solution and Water, dried over sodiumsulfate and evaporated to dryness in vacuo. The crude residue .(880 mg.)is crystallized from acetone and a little hexane furnishing pure16-keto-A -testololactone (700 mg.) possessing the following properties:MP. 262- 264; [@11 -120 (c, .40 in chlf. M51, 242 m (e =17,400); :21?5.74, shoulder at 5.78,

6.03, 6.18 and 6.26

Analysis.Calcd. for c,,H,,o, (314.36): 0, 72.59; H, 7.05. Found: 0,72,71; H, 7.19.

What is claimed is:

1. A compound selected from the group consisting of 16 keto-A-testololactone, 16oc-hydroxy-A -testololactone and an ester of thelatter with an unsubstituted carboxylic acid having less than ten carbonatoms selected from the group consisting of lower alkanoic acids,monocyclic aryl carboxylic acids, monocyclic aryl lower alkanoiccarboxylic acids, cycloalkane carboxylic acids and cycloalkenecarboxylic acids.

2. 16a hydroxy-A -testololactone.

3. 16u-hydroxy-A -testololactone acetate.

4. 16-keto-A -testololactone.

References Cited in the file of this patent UNITED STATES PATENTS2,755,289 Picka July 17, 1956 2,946,807 Fried et a1 July 26, 19602,982,693 Goodman et al May 2, 1961 2,991,230 Kita July .4, 19613,005,829 Wendler Oct. 24, 1961 FOREIGN PATENTS 792,803 Great BritainApr. 2, 1958

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF16-KETO-$1-TESTOLOLACTONE, 16A-HYROXY-$1-TESTOLOLACTONE AND AN ESTER OFTHE LATTER WITH AN UNSUBSTITUTED CARBOXYLIC ACID HAVING LESS THAN TENCARBON ATOMS SELECTED FROM THE GROUP CONSISTING OF LOWER ALKANOIC ACIDS,MONOCYCLIC ARYL CARBOXYLIC ACIDS, MONOCYCLIC ARYL LOWER ALKANOICCARBOXYLIC ACIDS, CYCLOALKANE CARBOXYLIC ACIDS AND CYCLOALKENECARBOXYLIC ACIDS.